Sesame plants, generally known as sesame (Sesamum indicum), belong to family Pedaliaceae and genus Sesamum. Sesame is native to central Africa and is the oldest cultivated oil plant with the history of over 6000 years. Sesame has been cultivated in various parts of the world.
Sesame seeds contain about 50% lipid and about 20% protein, in addition to various vitamins including vitamin B1, B2, and E. The main lipid component of sesame seed is triglyceride whose chief constituents are oleic acid and linoleic acid. Sesame also contains secondary metabolites generally known as sesamin, sesamolin, and lignan, which are characteristic components of the sesame plant.
Previous research has revealed various physiological activities of sesamin, which are found to be effective in improving cholesterol metabolism, and liver and immune functions (see Publication 1, for example). A separation and purification method of sesamin from the sesame seeds or wrung residues of sesame seeds has already been put to actual applications (see Patent Publications 1 and 2, for example). Sesamin is also commercially available as a medicament that enhances the liver function by promoting alcohol metabolism, etc. Other than sesamin, sesame lignans (sesaminol, sesamolin, etc.) have also been reported to have various physiological activities (see Publication 2, for example).
There has been some study on the biosynthesis of lignan (see Publication 3, for example). FIG. 1 is a schematic representation of a common biosynthesis pathway of lignan. The lignan is synthesized from a phenyl propanoid compound used as a starting material. In plants, lignan is believed to play role in the defense mechanism. As illustrated in FIG. 1, polymerization of conipheryl alcohol yields pinoresinol as the “first” lignan in the biosynthesis pathway. From pinoresinol, a wide variety of lignans are synthesized through distinct biosynthesis pathways of different plant species.
In the biosynthesis of sesamin, as shown in FIG. 1, piperitol is synthesized by the enzymatic action of a piperitol synthetase catalyzing (+)-pinoresinol, forming a methylene dioxybridge (circled in FIG. 1). Sesamin is synthesized by a sesamin synthetase forming another methylene dioxybridge in the piperitol. Experiments using membrane fractions of sesame seeds have shown that the enzymes catalyzing these reactions were two different kinds of cytochromes P450 (see Publication 4, for example).
The formation of methylene dioxybridge is often seen in the biosynthesis of alkaloid or flavonoid. For example, membrane fractions from cultured cells of Eschscholtzia calfornica are known to include cytochromes P450 that catalyze the biosynthesis of (S)-cheilanthifoline from (S)-scoulerine, and (S)-stylopine from (S)-cheilanthifoline, by forming a methylene dioxybridge in these compounds (see Publication 5, for example).
There have also been reported that membrane fractions from cultured cells of chickpea (Cicer arietinum) contain enzymes that catalyze the synthesis of Pseudobaptigenin and 5′-hydroxy Pseudobaptigenin produced by formation of a methylene dioxybridge in calycosin and pratensein, respectively. These enzymes have been identified as cytochromes P450 (see Publication 6, for example).
Others report the possibility that the deoxypodophyllotoxin 6-hydroxylase in cultured cells of Linum flavum may be cytochrome P450 (see Publication 7, for example).
The cytochrome P450 has also been found as an enzyme involved in the biosynthesis of berberine, which is a benzylisoquinoline alkaloid, in which (S)-tetrahydroberberine is synthesized by forming a methylene dioxybridge in (S)-tetrahydrocolimbamine. A gene that encodes this enzyme has been cloned from Coptis japonica (see Publication 8, for example).
The cytochromes P450 that catalyze various types of reactions as above comprise a superfamily of diverse molecular species, which are categorized based on the homology of their amino acid sequences. Different molecular species of cytochrome P450 belong to the same family if their identity is 40% or greater, and to the same sub family if their identity is 55% or greater. In the notation used in this classification, numbers denote family, and alphabets denote sub family (see Publication 9, for example). The tree diagram shown in FIG. 3 represents families and their interrelations. For example, the cytochrome P450 involved in the biosynthesis of berberine has been categorized as “CYP719.”
A single plant species include several hundred molecular species of cytochrome P450. However, as shown in FIG. 4, only a few of them have been identified based on their biochemical and physiological functions.
As a sesame-derived cytochrome P450, a gene (AY065995) that encodes p-coumarate 3-hydroxylase has been cloned, though it does not directly relate to the synthesis of sesamin or piperitol concerning the present invention.
Cloning of cytochrome P450 genes and their functional analysis have also been reported for various species of other organisms, as noted below.
For example, some of the cloned genes include:
A gene coding for petunia-derived flavonoid 3′,5′ hydroxylase (F3′,5′H) (see Publication 10, for example);
A gene coding for flavonoid 3′-hydroxylase (F3′H: CYP75B) (see Publication 11, for example);
A gene coding for sweetroot-derived (2S)-flavanon 2-hydroxylase (F2H: CYP93B1) (see Publication 12, for example);
A gene coding for 2-hydroxy-isoflavanon synthetase (IFS: CYP93C2) (see Publication 13, for example); and
A gene coding for isoflavon 2′-hydroxylase (12′H: CYP81E1) (see Publication 14, for example).
In addition, a gene coding for flavon synthetase II (FNSII: CYP93B3) has also been cloned from Antirrhinum majus, using 12′H (see Publication 15, for example).
Amino acid sequences of various cytochromes P450 that belong to the CYP81 family are found in Nelson, DR, The Cytochrome P450 Homepage, Human Genomics 4, 59-65. Among different functions of these cytochromes P450, Helianthus tuberosus-derived CYP81B1 is known to catalyze the hydrogenation of fatty acids (see Publication 16, for example). It is also known that the enzyme 12′H belongs to CPY81E.
However, none of these cytochromes P450 is involved in the formation of methylene dioxybridge.
As a gene that encodes an enzyme involved in the biosynthesis of lignan, a gene encoding a dirigent protein involved in the synthesis of pinoresinol in Forsythia intermedia has been reported (see Publication 17, and Patent Publication 3, for example). There have also been reports on a gene encoding a Pinoresinol-Lariciresinol reductase in Forsythia intermedia (see Publication 18, and Patent Publication 3, for example), and a gene encoding a Pinoresinol-Lariciresinol reductase in Thuja plicata (see Publication 19, for example). In other reports, a recombinant secoiso lariciresinol dehydrogenase and its use are discussed (see Publication 20, for example).
[Patent Publication 1]
Japanese laid-open publication No. 139579/2001 (published on May 22, 2001)
[Patent Publication 2]
Japanese laid-open publication No. 7676/1998 (published on Jan. 13, 1998)
[Patent Publication 3]
Japanese PCT laid-open publication No. 507931/2001 (published on Jun. 19, 2001)
[Patent Publication 4]
Japanese PCT laid-open publication No. 512790/2002 (published on May 8, 2002)
[Publication 1]
Mitsuo, Namiki, “Sesame, science and its functions,” published by Maruzen Planet
[Publication 2]
Publication of Japan Society for Bioscience, Biotechnology, and Agrochemistry, 76 805-813 2002
[Publication 3]
Lignans: bio synthesis and function, Comprehensive natural products chemistry vol. 1. 640-713, 1999
[Publication 4]
Phytochemisity, 49, 387, 1998
[Publication 5]
Phytochemisity, 30, 2953, 1991
[Publication 6]
Phytochemisity, 41, 457, 1996
[Publication 7]
Planta, 214, 288, 2001
[Publication 8]
J Biol Chem. 2003, May 5 [Epub ahead of print]
[Publication 9]
Nelson et al. Pharmacogenetics 6, 1-42, 1996
[Publication 10]
Nature 366 276-279, 1993
[Publication 11]
Plant J. 19, 441-451, 1999
[Publication 12]
FEBS Letters, 431, 287, 1998
[Publication 13]
Plant Physiology, 121, 821, 1999
[Publication 14]
Biochemical and Biophysical Research Communications, 251, 67, 1998
[Publication 15]
Plant and Cell Physiology, 40, 1182, 1999
[Publication 16]
J. Biol. Chem. 273, 7260, 1998
[Publication 17]
Plant Physiol. 123, 453, 2000
[Publication 18]
J. Biol. Chem. 271, 29473, 1996
[Publication 19]
J. Biol. Chem. 271, 618, 1999
[Publication 20]
J. Biol. Chem. 2001 Apr. 20; 276(16):12614-23, Epub 2001 Jan. 18
As described above, sesamin has a variety of physiological activities, which are known to be effective in improving various deficiencies. However, conventional sesamin production solely relied on a method using sesame seeds alone. In other words, sesamin production is completely dependent on sesame seeds. Consequently, it has been difficult to improve productivity or reduce the cost of sesamin production.
The problem can be solved effectively by genetic engineering techniques. However, to this date, no enzyme has been purified for the two kinds of cytochromes P450 that are known to be involved in the biosynthesis of sesamin in sesame seeds, and no gene for encoding these enzymes has been cloned. The situation is the same for the cytochromes P450 that form the methylene dioxybridge in different species of other organisms, or for the other kinds of cytochromes P450. There have been cloned genes encoding enzymes involved in the biosynthesis of lignan in different species of other organisms. However, none of them is involved in the synthesis of sesamin and/or piperitol.
Some of the genes derived from sesame seeds have been cloned, examples of which include AF240004, AF240005, and AF240006 encoding globulins as deposit proteins of seeds. Other examples include genes or enzymes involved in the synthesis or storage of lipids, including oleosin (J. Biochem. 122: 819-24, 1997), an acyl carrier protein desaturase (Plant Cell Physiol. 1996, 37, 201-5), Steroleosin (Plant Physiol. 2002, 128: 1200-11), and a fatty acid unsaturase (Plant Sci. 161 935-941 (2001)). However, none of these genes or enzymes is involved in the synthesis of lignan.
In other words, there has not been found a gene that encodes an enzyme involved in the synthesis of lignan. Accordingly, there is a strong need for identification of such enzymes, and genes encoding these enzymes.
The present invention was made in view of the foregoing problems, and an object of the invention is to provide a producing method of sesamin and/or piperitol with a gene encoding a sesame-derived enzyme, using, for example, recombinant organisms. The object is achieved by identifying a gene encoding an enzyme that catalyzes the formation of methylene dioxybridge between the hydroxyl group and methyl group of lignan, or more preferably a gene encoding an enzyme that catalyzes a reaction forming piperitol from pinoresinol, and/or a reaction forming sesamin from piperitol.